Systems and methods for illuminating and imaging objects

ABSTRACT

An illumination system includes a surface configured to have an imaging target placed thereon, a light source, a beam splitter and at least a first mirror. The beam splitter is configured to split the beam of light from the light source and the first mirror is configured to reflect a first beam from the beam splitter onto the surface with the imaging target. An imaging system includes an imaging surface configured to have an imaging target placed thereon, a mirror, and a capturing device. The capturing device is configured to capture an image of the imaging target through a path of emitted light that extends from the imaging target, reflects off of the mirror, and to the capturing device. The mirror, the capturing device, or both are configured to move in a diagonal direction with respect to the imaging surface to reduce a length of the path of emitted light. Systems and methods to calibrate an imaging system to remove or reduce non-uniformities within images of samples due to imaging system properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/508,747 filed May 19, 2017, and U.S.Provisional Patent Application No. 62/408,018 filed Oct. 13, 2016, andU.S. Provisional Patent Application No. 62/408,006 filed Oct. 13, 2016,each of which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure is directed to devices, systems, and methods forilluminating an object and obtaining high resolution images of theobject. The present disclosure is also related to methods for imagenon-uniformity correction.

BACKGROUND

There is a need for imaging devices, systems, and methods that providehigh resolution images of an object that do not rely on approaches suchas digital magnification or use of a zoom lens. Digital magnificationcan often lead to image pixilation as an image is magnified. The use ofa zoom lens is difficult to implement in many circumstances as theability to satisfy various requirements such as large aperture, focallength, working distance, distortion, field curvature, and signalattenuation in a robust manner is often difficult.

There is also a need for illumination devices, systems, and methods thatcan provide two or more beams of light to illuminate an imaging target,particularly in a uniform illumination approach, without the use of twoor more light sources at the same time. The use of multiple lightsources often leads to the multiple beams of light of differing opticalpower being applied to the imaging target given the use of two lightsources that have to be maintained separately and may have had differentoptical properties after manufacturing or as configured within thedevice or system. Furthermore, the use of multiple light sources oftenleads to greater non-uniformity of the overall illumination of theimaging target and also greater mechanical complexity of theillumination system, which in turn increases maintenance requirementsand increases the likelihood of non-uniform illumination. Another commonproblem during imaging (irrespective of imaging mode) is imagenon-uniformity. For example, when identical samples are placed atdifferent locations of an imaging surface or a field of view, thecorresponding image appears to be non-uniform based on the location,even though the identical samples emits identical signal. There is aneed in the art to address image non-uniformity.

SUMMARY

An illumination system is disclosed. The illumination system includes asurface, a light source, a beam splitter, a first mirror, and a secondmirror. The surface is configured to have an imaging target placedthereon. The light source is configured to emit a beam of light. Thebeam splitter is configured to split the beam of light from the lightsource into a first beam and a second beam. The first mirror isconfigured to reflect the first beam to provide a reflected first beamthat illuminates the surface. The second mirror is configured to reflectthe second beam to provide a reflected second beam that illuminates thesurface.

In another embodiment, the illumination system includes a surface, alight source, a beam splitter, and a first mirror. The surface isconfigured to have an imaging target placed thereon. The light source isconfigured to emit a beam of light. The beam splitter is configured tosplit the beam of light from the light source into a first beam and asecond beam. The second beam illuminates the surface. The first mirroris configured to reflect the first beam from the beam splitter toprovide a reflected first beam that illuminates the surface.

An illumination method is also disclosed. The method includes providinga surface with an imaging target placed thereon. The method alsoincludes providing a beam of light with a light source. The methodfurther includes splitting the beam of light into a first beam and asecond beam. The method further includes illuminating the surface.Illuminating includes: (i) using a first mirror to reflect the firstbeam to produce a reflected first beam that illuminates the surface, and(ii) using a second mirror to reflect the second beam to produce areflected second beam that illuminates the surface.

In another embodiment, the illumination method includes providing a beamof light with a light source. The method also includes splitting thebeam of light into a first beam and a second beam. The method furtherincludes illuminating a surface with an imaging target placed thereon.Illuminating includes using a first mirror to reflect the first beam toproduce a reflected first beam that illuminates the surface. The secondbeam is split from the beam of light such that it illuminates thesurface.

An imaging system is also disclosed. The imaging system includes animaging surface, a mirror, and a capturing device. The imaging surfaceis configured to have an imaging target placed thereon. The capturingdevice is configured to capture an image of the imaging target through apath of emitted light that extends from the imaging target, reflects offof the mirror, and to the capturing device. The mirror, the capturingdevice, or both are configured to move in a diagonal direction withrespect to the imaging surface to reduce a length of the path of emittedlight.

In another embodiment, the imaging system includes an imaging surface, amirror, a mirror shaft, a capturing device, a capturing device shaft,and a transmission block. The imaging surface is configured to have animaging target placed thereon. The mirror is configured to move in afirst diagonal direction along the mirror shaft. The capturing device isconfigured to capture an image of the imaging target through a path ofemitted light that extends from the imaging target, reflects off of themirror, and to the capturing device. The capturing device is configuredto move in a second diagonal direction along the capturing device shaft.The transmission block transmits movement between the mirror and thecapturing device, thereby causing the mirror and the capturing device tomove simultaneously.

An imaging method is also disclosed. The method includes placing animaging target on an imaging surface. The method also includes causing acapturing device, a mirror, or both to move in a diagonal direction withrespect to the imaging surface. The method further includes capturing animage of the imaging target, using the capturing device, through a pathof emitted light that extends from the imaging target, reflects off ofthe mirror, and to the capturing device.

An illumination and imaging system is also disclosed. The systemincludes a surface configured to have an imaging target placed thereon.A light source is configured to emit a beam of light. A beam splitter isconfigured to split the beam of light from the light source into a firstbeam and a second beam. A first illumination mirror is configured toreflect the first beam to provide a reflected first beam thatilluminates the surface. A second illumination mirror is configured toreflect the second beam to provide a reflected second beam thatilluminates the surface. A capturing device is configured to capture animage of the imaging target through a path that extends from the imagingtarget, reflects off of an emission mirror, and to the capturing device.The emission mirror, the capturing device, or both are configured tomove in a diagonal direction with respect to the surface to reduce alength of the path.

An illumination and imaging method is also disclosed. The methodincludes placing an imaging target on a surface. The method alsoincludes emitting a beam of light from a light source. The methodfurther includes splitting the beam of light into a first beam and asecond beam. The method further includes illuminating the imagingtarget. Illuminating includes: (i) using a first illumination mirror toreflect the first beam to produce a reflected first beam thatilluminates the surface, and (ii) using a second illumination mirror toreflect the second beam to produce a reflected second beam thatilluminates the surface. The method further includes capturing an imageof the imaging target, using a capturing device, through a path thatextends from the imaging target, reflects off of an emission mirror, andto the capturing device.

The disclosure, in some embodiments, describes methods for generating animage corrected for a non-uniformity. In some embodiments, anon-uniformity is displayed as images with signals of varying intensityfor an identical signal measured at different locations on the field ofview.

A non-uniformity correction method of the present disclosure can beapplied to images obtained from a variety of samples, includingbiological samples that comprise biological molecules such as proteins,peptides, glycoproteins, modified proteins, nucleic acids, DNA, RNA,carbohydrates, lipids, lipidoglycans, biopolymers and other metabolitesgenerated from cells and tissues and combinations thereof. A bimoleculeor biological sample having a biomolecule can be imaged alone or can beimaged while it is dispersed, located or embedded in a membrane, a gel,a filter paper, slide glass, microplate, or a matrix, such as apolyacrylamide gel or nitrocellulose or PDVF membrane blot, an agarosegel, an agar plate, a cell culture plate or a tissue section slide. Anon-uniformity correction method of the present disclosure can beapplied to images obtained from any of the samples described above.

A non-uniformity correction method of the present disclosure can beapplied to an image generated by a chemiluminescence change to abiological sample or to an image generated by a fluorescence change tothe sample. A non-uniformity correction method of the present disclosurecan be applied to an image generated by bioluminescent imaging,transillumination or reflective light imaging.

In one embodiment, a method for generating an image corrected for anon-uniformity comprises: calculating a relative illumination of animaging lens for a plurality of pixels on an imaging sensor; generatinga flat fielding matrix based upon the relative illumination; capturingor acquiring an image of one or more biological samples, wherein theimage has a non-uniformity; and adjusting the captured image with theflat fielding matrix to generate an image corrected for thenon-uniformity.

In one embodiment, generating a flat fielding matrix comprises invertingrelative illumination to generate a flat fielding matrix. In someembodiments, relative illumination is calculated using an equationobtained by a linear or a non-linear curve fitting regression. The curvecan be a first degree polynomial, a second degree polynomial, a thirddegree polynomial, or the like. Calculations of flat fielding matrixgenerate a flat fielding matrix value.

Adjusting the captured image with the flat fielding matrix comprisesmultiplying the captured or acquired image of one or more biologicalsamples by the value of flat fielding matrix. In some embodiments,adjusting the captured or acquired image with the flat fielding matrixfurther comprises multiplying the captured or acquired image of the oneor more biological samples by the value of the flat fielding matrix on apixel-to-pixel basis to generate a flat fielded image. In someembodiments, the flat fielded image displays a correct ratio of a signallevel of each captured or acquired image of the one or more biologicalsample irrespective of its location on a field of view.

In one embodiment, the present disclosure provides a method forgenerating an image corrected for a non-uniformity, comprising:calculating a relative illumination of an imaging lens of a plurality ofpixels on an imaging sensor; inverting the relative illumination togenerate a flat fielding matrix; capturing or acquiring an image of abiological sample; and multiplying the captured or acquired image of abiological sample by the value of the flat fielding matrix on apixel-to-pixel basis to generate a flat fielded image.

In one embodiment, the disclosure describes methods for generating animage corrected for a non-uniformity, comprising: capturing or acquiringan image of one or more biological samples, wherein the image has anon-uniformity; and adjusting the captured or acquired image with a flatfielding matrix to generate an image corrected for the non-uniformity.Adjusting the captured or acquired image can comprise multiplying thecaptured image of the one or more biological samples by the value of theflat fielding matrix on a pixel-to-pixel basis to generate a flatfielded image.

In some embodiments, the flat fielding matrix is in an imager or imagingsystem. The flat fielding matrix is available to a user using theimager. The flat fielding matrix value can be stored in an imagingdevice. In some embodiments, the flat fielding matrix value can bestored in a software component or computer component of the imagingdevice. The flat fielding matrix value is available to a user using theimaging system performing the non-uniformity correction. In someembodiments, the flat fielding matrix is a flat fielding master matrix.

In one embodiment, a method for generating a flat fielding matrix forcorrecting images for a non-uniformity, comprises: calculating arelative illumination of an imaging lens for a plurality of pixels on animaging sensor; generating a flat fielding matrix based upon therelative illumination and normalizing flat fielding matrix based on themaximum pixel intensity value in the matrix. In one embodiment,generating the flat fielding matrix comprises inverting the relativeillumination and normalization of the values in the matrix. For example,a manufacturer or a user can generate a flat fielding matrix for futureuse.

Additional objects and advantages of the disclosure will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or can be learned by practice of the disclosure. Theobjects and advantages of the disclosure will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate the present disclosure andtogether with the description, serve to explain the principles of thepresent disclosure.

FIGS. 1 and 2 illustrate perspective views (from different angles) of animaging system, according to an embodiment.

FIGS. 3 and 4 illustrate perspective views of the imaging system withsome of the components from FIGS. 1 and 2 omitted to better show theshafts to which the components are coupled, according to an embodiment.

FIGS. 5, 6, 7, and 8 illustrate cross-sectional side views of theimaging system proceeding through increasing levels of zoom, accordingto an embodiment.

FIG. 9 illustrates a beam of light being emitted from an illuminationmodule of the imaging system, according to an embodiment.

FIG. 10 illustrates the epi-illumination beam being at least partiallyobstructed by a mirror when the center of the path of emitted lightremains centered on the mirror as the mirror moves during zooming,according to an embodiment.

FIG. 11 illustrates a simplified schematic side view of the beam oflight being emitted from the illumination module of the imaging systemshown in FIG. 9 , according to an embodiment.

FIG. 12 illustrates a simplified schematic top view of the beam of lightbeing emitted from an illumination module with additional mirrors,according to an embodiment.

FIG. 13 illustrates a simplified schematic top view of the beam of lightbeing emitted from an illumination module with two beam splitters,according to an embodiment.

FIG. 14 illustrates a cross-sectional side view of the beam of lightpassing through an aperture before reaching the beam splitter, accordingto an embodiment.

FIG. 15 illustrates a perspective view of a luminometer reference plate.

FIG. 16A illustrates the stacked images of one light spot of theluminometer reference plate in different locations on the field of view,and FIG. 16B illustrates a graph showing the signal of the same spot indifferent locations.

FIG. 17 illustrates a graph showing relative illumination of an imaginglens within an imaging system and best fit result from a non-linearregression, according to an embodiment.

FIG. 18A illustrates the modified image of FIG. 16A with application ofthe flat fielding master matrix, and FIG. 18B illustrates a graphshowing a ratio of intensity of spots over the spot of maximum intensityin FIG. 18A, according to an embodiment.

FIG. 19A illustrates an image of a chemiluminescence sample at 1× zoomlevel in the middle of the field of view, FIG. 19B illustrates an imageof the chemiluminescence sample at 1× zoom level at a position betweenthe middle and the top right diagonal position of the field of view,according to an embodiment, and FIG. 19C illustrates an image of thechemiluminescence sample at 1× zoom level at the top right diagonalposition of the field of view.

FIG. 20A illustrates an image showing two rows of bands that arequantified, FIG. 20B illustrates a graph showing the intensity of thebands of FIG. 20A on the first row before flat fielding, FIG. 20Cillustrates a graph showing the intensity of the bands on the first rowof FIG. 20A after flat fielding, FIG. 20D illustrates a graph showingthe intensity of the bands on the second row of FIG. 20A before flatfielding, and FIG. 20E illustrates a graph showing the intensity of thebands on the second row of FIG. 20A after flat fielding, according to anembodiment.

FIG. 21A illustrates a table showing relative illumination of an imaginglens against sensor image height with a 1× zoom, and FIG. 21Billustrates a table showing relative illumination of an imaging lensagainst sensor image height with a 2× zoom, according to an embodiment.

FIG. 22 illustrates a plot showing that relative illumination issymmetrical with respect to the center of the CCD, according to anembodiment.

FIG. 23A illustrates a graph showing a best fit non-linear regressioncurve with a 1× zoom, and FIG. 23B illustrates a graph showing a bestfit non-linear regression curve with a 2× zoom, according to anembodiment.

FIG. 24 illustrates a simulation image at a 1× zoom, according to anembodiment.

FIG. 25 illustrates a flat fielding master image, according to anembodiment.

FIGS. 26A, 26B, and 26C are images after applying flat fielding. FIG.26A illustrates an image of in a middle position of the field of viewwith a 1× zoom, FIG. 26B illustrates an image in a position between themiddle position and the top right diagonal position of the field of viewwith a 1× zoom, according to an embodiment, and FIG. 26C illustrates animage of in a top right diagonal position of the field of view with a 1×zoom.

FIG. 27 illustrates an image showing two rows of eight bands each,according to an embodiment.

FIG. 28A illustrates a graph showing the first row of FIG. 27 taken at1× zoom before flat fielding, FIG. 28B illustrates a graph showing thefirst row of FIG. 27 taken at 1× zoom after flat fielding, FIG. 28Cillustrates a graph showing the second row of FIG. 27 taken at 1× zoombefore flat fielding, and FIG. 28D illustrates a graph showing thesecond row of FIG. 27 taken at 1× zoom after flat fielding at, accordingto an embodiment.

FIG. 29A illustrates a graph showing the first row of FIG. 27 taken at2× zoom before flat fielding, FIG. 29B illustrates a graph showing thefirst row of FIG. 27 taken at 2× zoom after flat fielding, FIG. 29Cillustrates a graph showing the second row of FIG. 27 taken at 2× zoombefore flat fielding, and FIG. 29D illustrates a graph showing thesecond row of FIG. 27 taken at 2× zoom after flat fielding, according toan embodiment.

FIG. 30 illustrates a chart showing the membrane position (e.g., middle,top right) before and after flat fielding, according to an embodiment.

DETAILED DESCRIPTION

Reference will now be made in detail to exemplary implementations of thepresent disclosure, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same reference numberswill be used throughout the drawings to refer to the same or like parts.In the following description, reference is made to the accompanyingdrawings that form a part thereof, and in which is shown by way ofillustration specific exemplary implementations in which the presentdisclosure can be practiced. These implementations are described insufficient detail to enable those skilled in the art to practice thepresent disclosure and it is to be understood that other implementationscan be utilized and that changes can be made without departing from thescope of the present disclosure. The following description is,therefore, merely exemplary.

FIGS. 1 and 2 illustrate perspective views of portions of an imagingsystem 100 taken from different angles, according to an embodiment. Theimaging system 100 may include an imaging surface 110. In one example,the imaging surface 110 may be or include a tray or a screen. Theimaging surface 110 may be planar and substantially horizontal (i.e.,parallel with the ground). An imaging target 112 may be placed on theimaging surface 110. The imaging target 112 may be or include biologicalmaterials such as nucleic acids and/or proteins associated withpolyacrylamide gels, agarose gels, nitrocellulose membranes, and PVDFmembranes. The imaging target 112 may also be or include non-biologicalmaterials such as manufactured articles and documents.

The imaging system 100 may also include a mirror 120. The mirror 120 maybe positioned (e.g., directly) above the imaging surface 110 and theimaging target 112. The mirror 120 may include a reflective surface. Asshown, the reflective surface may be planar; however, in otherembodiments, the reflective surface may be curved. When the reflectivesurface of the mirror 120 is planar, the reflective surface of themirror 120 may be oriented at an angle with respect to the imagingsurface 110 (i.e., with respect to horizontal). The angle may be fromabout 10° to about 80°, about 20° to about 70°, or about 30° to about60°. For example, the angle may be about 45°.

The imaging system 100 may also include a capturing device 130. Thecapturing device 130 may include a detector housing 140, one or morefilters (one is shown: 150), and a camera 160. The detector housing 140may be positioned above the imaging surface 110 and laterally (e.g.,horizontally) offset from the mirror 120. The detector housing 140 mayinclude a lens 142. The detector housing 140 may also include a filterwheel, a motor, and/or sensors that control the focus and aperture ofthe lens 142. The lens 142 may be planar, and a central longitudinalaxis through the lens 142 may intersect the reflective surface of themirror 120. As such, a path of emitted light may extend verticallybetween the imaging target 112 and the mirror 120, and laterally betweenthe mirror 120 and the lens 142 of the detector housing 140. As usedherein, a “path of emitted light” refers to a route of a field of viewfrom an imaging target 112 to the camera 160 through the lens 142.

The filter 150 may be coupled to and positioned behind the detectorhousing 140, and the path of emitted light may extend through thedetector housing 140 and into the filter 150. The filter 150 may be anelectromagnetic (“EM”) filter that transmits only selected wavelengthsof light to the camera 160. Placing the filter 150 behind the lens 142may allow the filter 150 to be smaller than if the filter 150 is placedin front of the lens 142. Both excitation and emission light may enterthe lens 142. The excitation light blocked by the filter 150 may hit thelens 142 and surrounding surfaces, and a certain amount of theexcitation light may bounce back to the filter 150 again and may passthrough the filter 150 this time. In another embodiment, a filter may beplaced in front of the lens 142. Because excitation is blocked by thefilter in front of the lens 142, there may be very little excitationlight after the filter e.g., almost no excitation light propagatesinside the lens 142 to the camera 160, which makes stray light controleasy and the background signal lower. The filter in front of the lens142 may be larger than the filter 150 behind the lens. Therefore, thesize of the filter wheel may be larger and occupy more space. In certainembodiments, a second filter may also be placed in front of lens 142. Insuch embodiments, the second filter, which may be a notch filter incertain embodiments, is placed in front of lens 142 while filter 150 isplaced behind lens 142. These embodiments can provide an advantage ofthe two filters working together to minimize stray light, includingstray excitation light, from affecting the emissions captured by camera160.

The camera 160 may be coupled to and positioned behind the filter 150,and path of emitted light may extend through the filter 150 and into thecamera 160, where the camera 160 may capture one or more (e.g.,filtered) images of the imaging target 112.

The imaging system 100 may also include a first sensor 190 in a firstposition and a second sensor 192 in a second position (shown in FIG. 1). The first sensor 190 may be a limit sensor that is configured tolimit the travel distance of the detector housing 140, the filter 150and the camera 160. The second sensor 192 may be a homing sensor that isconfigured to set the detector housing 140, the filter 150 and thecamera 160 to the initial default position.

The imaging system 100 may also include an illumination module 200(shown in FIG. 1 ). The illumination module 200 may be or include anepi-illumination module and/or a diascopic illumination module. Theillumination module 200 may include a light source 210. The light source210 may be or include one or more light-emitting diodes (“LEDs”). Theillumination module 200 may also include an excitation filter 220 thatis coupled to and positioned in front of the light source 210. Theexcitation filter 220 may be configured to limit the range of wavelengthof light from the light source 210. The illumination module 200 may alsoinclude a lens 230 that is coupled to and positioned in front of theexcitation filter 220. In at least one embodiment, the lens 230 may beor include a toroidal lens. The illumination module 200 may also includea beam splitter 240 that is coupled to and positioned in front of thelens 230. The beam splitter 240 may be configured to split or divide thebeam from the light source 210 into two or more beam portions. Theillumination module 200 may also include a near-infrared (“NIR”)illumination module and mirror 250 that may be positioned proximate to(e.g., below) the light source 210, the excitation filter 220, the lens230, the beam splitter 240, or a combination thereof. The NIRillumination module and mirror 250 may include a LED that provides lightin the NIR range. The NIR illumination module and mirror 250 may alsoreflect the NIR light into the beam splitter 240 at substantially thesame angle as the visible light. The illumination module 200 may alsoinclude a back mirror 260 that is positioned below the capturing device130 and/or above the light source 210, the excitation filter 220, thelens 230, the beam splitter 240, or a combination thereof. Theillumination module 200 may also include a front mirror 262. The imagingsurface 110 may be positioned laterally (e.g., horizontally) between thelight source 210, the excitation filter 220, the lens 230, the beamsplitter 240 on one side and the front mirror 262 on the other side. Thefront mirror 262 may also be positioned above the imaging surface 110.Although not shown, the illumination module 200 may also include adiascopic illumination module and a light source (e.g., LEDs). The lightsource or light sources for diascopic illumination may be positionedbelow the imaging surface 110 to provide illumination through theimaging surface 110 and the imaging target 112.

FIGS. 3 and 4 illustrate perspective views of the imaging system 100with some of the components (e.g., the mirror 120 and the capturingdevice 130) omitted to better show the shafts 124, 134 to which thecomponents are coupled, according to an embodiment.

The mirror 120 (not shown in FIGS. 3 and 4 ) may be coupled to a mirrorsupport structure 122, and the capturing device 130 (also not shown inFIGS. 3 and 4 ) may be coupled to a capturing device support structure132. The mirror support structure 122 may be coupled to and configuredto slide back and forth along a mirror shaft 124 in an axial directionthat is aligned (e.g., parallel) with the mirror shaft 124. Thecapturing device support structure 132 may be coupled to and configuredto slide back and forth along a capturing device shaft 134 in an axialdirection that is aligned (e.g., parallel) with the capturing deviceshaft 134. A transmission block 180 may be coupled to and configured toslide back and forth along a transmission block shaft 184 in an axialdirection that is aligned (e.g., parallel) with the transmission blockshaft 184. In at least one embodiment, the mirror shaft 124, thecapturing device shaft 134, the transmission block shaft 184, or acombination thereof may be in a single plane.

The mirror shaft 124 may be oriented diagonally with respect to theupper surface of the imaging surface 110. As used herein “diagonally”refers to a direction that is neither parallel nor perpendicular to theimaging surface 110. More particularly, the mirror shaft 124 may beoriented at an angle with respect to the imaging surface 110 that isfrom about 10° to about 170°, about 40° to about 140°, or about 70° toabout 110° (when viewed from the direction shown in FIGS. 3 and 4 ). Forexample, the angle 126 may be about 91° (when viewed from the directionshown in FIGS. 3 and 4 ).

The capturing device shaft 134 may also be oriented diagonally withrespect to the imaging surface 110 (i.e., with respect to horizontal).More particularly, the capturing device shaft 134 may be oriented at anangle 136 with respect to the imaging surface 110 that is from about 10°to about 80°, about 20° to about 70°, or about 30° to about 60° (whenviewed from the direction shown in FIGS. 3 and 4 ). For example, theangle 136 may be about 35° (when viewed from the direction shown inFIGS. 3 and 4 ). An angle 127 between the mirror shaft 124 and thecapturing device shaft 134 may be from about 80° and about 140°, about90° and about 130°, or about 100° and about 120°. For example, the angle127 may be about 123°.

The transmission block shaft 184 may be positioned between the mirrorshaft 124 and the capturing device shaft 134 (i.e., within the angle127). The transmission block shaft 184 may also be oriented diagonallyor perpendicular (i.e., vertical) with respect to the upper surface ofthe imaging surface 110.

Referring to FIG. 4 , a first transmission shaft 138 may be coupled toand extend between the capturing device support structure 132 and thetransmission block 180. The capturing device support structure 132 (andthe capturing device 130), the transmission block 180, or a combinationthereof may be configured to slide axially along the first transmissionshaft 138. A second transmission shaft 128 may be coupled to and extendbetween the mirror support structure 122 and the transmission block 180.The mirror support structure 122 (and the mirror 120), the transmissionblock 180, or a combination thereof may be configured to slide axiallyalong the second transmission shaft 128.

The imaging system 100 may include one or more motors (one is shown inFIG. 3 : 170). The motor 170 may cause the mirror 120 and/or thecapturing device 130 (e.g., the detector housing 140, the filter 150,and the camera 160) to move with respect to the imaging surface 110 andthe imaging target 112. In the embodiment shown, the single motor 170may cause the mirror 120 and the capturing device 130 to movesimultaneously. This simultaneous movement with a single motor may beenabled by use of a power transmission shaft and block that links themirror 120 and the capturing device 130, such as the first transmissionshaft 138, the second transmission shaft 128, and the transmission block180 as described above in reference to FIG. 4 . Such an approachprovides the advantage of controlling the motion of both the mirror 120and the capturing device 130 with a single motor and in a synchronizedfashion that is not dependent on a separate control mechanism, such ascontrol software, thereby providing the advantage of lower complexityand cost, reduced maintenance requirements, and an improved ability tomaintain a consistent image center at different degrees of zoom. Inanother embodiment, a first motor may cause the mirror 120 to move, anda second motor may cause the capturing device 130 to move, and a ratioof the movement of the mirror 120 with respect to the capturing device130 may be fixed. Fixing this ratio of movement may be accomplished viathe software controlling the first and second motors and would enablesynchronized movement while also keeping the center of the imageconsistent during zooming. The transmission block 180 may be coupled tothe mirror 120 and the capturing device 130. When a single motor 170 isused, the transmission block 180 may link the movement of the mirror 120and the capturing device 130, as described in greater detail in FIGS. 3and 4 . In a different embodiment, one or more belt drives or otherdevices may be used to move the mirror 120 and the capturing device 130.

Referring again to FIGS. 3 and 4 , the motor 170 may be coupled to alead screw 172 via a coupler 174. The coupler 174 may transfer rotarymotion of the motor 170 to the lead screw 172, thereby causing the leadscrew 172 to rotate. The lead screw 172 may be parallel with thecapturing device shaft 134. When the lead screw 172 rotates in a firstdirection, the lead screw 172 may push the capturing device supportstructure 132 (and the capturing device 130) in a first axial directionalong the capturing device shaft 134. Conversely, when the lead screw172 rotates in a second (i.e., opposite) direction, the lead screw 172may pull the capturing device support structure 132 (and the capturingdevice 130) in a second (i.e., opposite) axial direction along thecapturing device shaft 134.

When the capturing device support structure 132 (and the capturingdevice 130) move in the first axial direction along the capturing deviceshaft 134, the first transmission shaft 138 may cause the transmissionblock 180 to move in a first axial direction along the transmissionblock shaft 184. Conversely, when the capturing device support structure132 (and the capturing device 130) move in the second axial directionalong the capturing device shaft 134, the first transmission shaft 184may cause the transmission block 180 to move in a second (i.e.,opposite) axial direction along the transmission block shaft 184.

When the transmission block 180 moves in the first axial direction alongthe transmission block shaft 184, the second transmission shaft 128 maycause the mirror support structure 122 (and the mirror 120) to move in afirst axial direction along the mirror shaft 124. Conversely, when thetransmission block 180 moves in the second axial direction along thetransmission block shaft 184, the second transmission shaft 128 maycause the mirror support structure 122 (and the mirror 120) to move in asecond (i.e., opposite) axial direction along the mirror shaft 124.

Thus, as will be appreciated, the mirror 120 and the capturing device130 may move together simultaneously. When the mirror 120 and thecapturing device 130 move in their respective first axial directions,the total length of the path of emitted light from the imaging target112 (reflecting off the mirror 120) to the lens 142 of the detectorhousing 140 may decrease, and when the mirror 120 and the capturingdevice 130 move in their respective second axial directions, the totallength of the path of emitted light from the imaging target 112(reflecting off the mirror 120) to the lens 142 of the detector housing140 may increase.

FIGS. 5, 6, 7 and 8 illustrate cross-sectional side views of the imagingsystem 100 proceeding through increasing levels of zoom, according to anembodiment. More particularly, FIG. 5 illustrates the imaging system 100with no zoom. The total length of a center of the path of emitted light115 from the imaging target 112 (reflecting off the mirror 120) to thelens 142 of the detector housing 140 when there is no zoom may be, forexample, about 455 mm in one embodiment, but the total length of thecenter of the path of emitted light 115 will depend on the overallconfiguration of a system and its components, including the propertiesof the capturing device 130. The path of emitted light 114 may contact afirst portion (e.g., surface area) of the mirror 120 when there is nozoom. The first portion (e.g., surface area) may be from about 50% toabout 100%, about 75% to about 99%, or about 85% to about 95% of thetotal surface area of the mirror 120.

Referring now to FIG. 6 , the capturing device 130 and the mirror 120may move in their respective first axial directions to reduce the totallength of the center of the path of emitted light 115 (i.e., to zoom inon the imaging target 112 on the imaging surface 110). The center of thepath of emitted light 115 between the imaging target 112 and the mirror120 may remain stationary as the mirror 120 moves diagonally (i.e., thevertical arrow is identical in FIGS. 5 and 6 ). As a result, the pointon the mirror 120 that the center of the path of emitted light 115contacts may vary/move as the mirror 120 and the capturing device 130move in their respective first axial directions. For example, the centerof the path of emitted light 115 may contact the mirror 120 at point116A in FIG. 5 and at point 116B in FIG. 6 . In addition, the portion(e.g., surface area) of the mirror 120 that the path of emitted light114 contacts may decrease as the mirror 120 and the capturing device 130move in their first axial directions.

Referring now to FIG. 7 , the mirror 120 and the capturing device 130may move further in their respective first axial directions to furtherreduce the total length of the center of the path of emitted light 115(i.e., to zoom in on the imaging target 112 on the imaging surface 110).The center of the path of emitted light 115 between the imaging target112 and the mirror 120 may remain stationary as the mirror 120 movesdiagonally (i.e., the vertical arrow is identical in FIGS. 5-7 ). As aresult, the point on the mirror 120 that the center of the path ofemitted light 114 contacts may vary/move as the mirror 120 and thecapturing device 130 move in their respective first axial directions.For example, the center of the path of emitted light 114 may contact themirror 120 at point 116C in FIG. 7 . In addition, the portion (e.g.,surface area) of the mirror 120 that the path of emitted light 114contacts may continue to decrease as the mirror 120 and the capturingdevice 130 move further in their first axial directions.

Referring now to FIG. 8 , the mirror 120 and the capturing device 130have minimized the total length of the center of the path of emittedlight 115 (i.e., to maximize zoom on the imaging target 112 on theimaging surface 110). The center of the path of emitted light 115between the imaging target 112 and the mirror 120 may remain stationaryas the mirror 120 moves diagonally (i.e., the vertical arrow isidentical in FIGS. 5-8 ). As a result, the point on the mirror 120 thatthe center of the path of emitted light 115 contacts may vary/move asthe mirror 120 and the capturing device 130 move in their respectivefirst axial directions. For example, the center of the path of emittedlight 115 may contact the mirror 120 at point 116D in FIG. 8 . In anexample, the total length of the center of the path of emitted light 115from the imaging target 112 (reflecting off the mirror 120) to the lens142 of the detector housing 140 when the zoom is maximized may be, forexample, about 215 mm. Thus, the imaging system 100 may be configured tozoom from about 1× to about 2×; however, in other embodiments, theimaging system 100 may be configured to zoom even further (i.e., greaterthan 2×). In addition, the portion (e.g., surface area) of the mirror120 that the path of emitted light 114 contacts may decrease as the zoomincreases. For example, the portion (e.g., surface area) may be fromabout 5% to about 80%, about 10% to about 70%, or about 20% to about 60%of the total surface area of the mirror 120 when the zoom is maximized.

FIG. 9 illustrates a beam of light (e.g., an epi-illumination beam) 212being emitted from the illumination module 200, according to anembodiment. The beam of light 212 may be emitted from the light source210 (see FIG. 1 ) of the illumination module 200. The beam of light 212may be split into first and second beams 213, 214 by the beam splitter240. The first beam 213 may reflect off of the back mirror 260 andilluminate the imaging target 112, and the second beam 214 may reflectoff of the front mirror 262 and illuminate the imaging target 112. Thisis described in greater detail below with respect to FIG. 11 . Inanother embodiment, the beam of light 212 may be emitted from the NIRillumination module 250 and reflected off of the mirror in the NIRillumination module 250 to the imaging target 112. In at least oneembodiment, the beam of light 212 may extend through the path of emittedlight 114 to illuminate the imaging target 112, which may reflect theillumination light or may contain fluorescent components that emit lightafter excitation with the epi-illumination.

When the mirror 120 and the capturing device 130 are at their positionsof maximum zoom, as shown in FIG. 9 , a lower end 139 of the capturingdevice 130 may be positioned below a lower end 129 of the mirror 120. Asa result, the mirror 120 may not obstruct the beam of light 214 at anypoint along the mirror shaft 124.

FIG. 10 illustrates the epi-illumination beam 212 being at leastpartially obstructed by the mirror 120, according to an embodiment. Ifthe center of the path of emitted light 114 remains fixed on the samepoint on the mirror 120 as the mirror moves (e.g., point 116A from FIG.5 ), the lower end 129 of the mirror 120 may be positioned below thelower end 139 of the capturing device 130 when the capturing device 130and the mirror 120 are at their positions of maximum zoom. As a result,the mirror 120 may at least partially obstruct the beam of light 212.Thus, as shown in FIGS. 5-9 , the center of the path of emitted light114 may move/vary on the mirror 120 as the mirror 120 moves duringzooming to avoid blocking the beam of light 212.

Epi-illumination and/or excitation may be used for a fluorescence modeof protein analysis. Many fluorescent dyes may be used for protein stainand/or western blot, and different dyes have different excitationspectrum profiles, and thus need different colors of excitation light. Acertain excitation power may provide a fluorescence imaging signalwithin an acceptable imaging exposure time. If the illumination and/orexcitation power varies too much across the field of view, there may beone or more dark areas where it is difficult to see the land/band of thesample, or the land/band may be seen in the dark area(s), but the signalin the brighter areas becomes saturated. As a result, substantiallyuniform illumination may improve imaging quality.

There are two types of epi-illumination: on-axis and off-axis (i.e.,oblique). On-axis illumination may generate bright spots on the imagedue to certain light reflections from the sample. Off-axis illuminationis one way to remedy this problem. In some embodiments, the off-axisangle may be greater than or equal to a predetermined amount to avoidgenerating the bright spot.

FIG. 11 illustrates a simplified schematic side view of the beam oflight 212 being emitted from the illumination module 200 shown in FIG. 9, according to an embodiment. The beam of light 212 may be emitted fromthe light source 210 (see FIG. 1 ) of the illumination module 200. Thelight source 210 may include a first LED for fluorescent excitationand/or a second LED for near IR. In another embodiment, a tungstenhalogen lamp may be used to cover both spectrums. For any particularchannel, there may be only a single beam of light. The light source 210may have a single color. In at least one embodiment, the light source210 may be a white light source, and optical filters may be used togenerate different colors.

The beam of light 212 may be split into a first beam 213 and a secondbeam 214 by the beam splitter 240. Although not shown, in otherembodiments, the beam splitter 240 may be configured to split the beamof light 212 into three or more beams. As used herein, the term “beamsplitter” includes one or more optical components capable of splittingor otherwise separating a beam of light, and includes but is not limitedto prisms, plates, dielectric mirrors, metal coated mirrors, beamsplitter cubes, fiber optic beam splitters, and optical fibersconfigured to collimate light into a bundle before producing two or moreoutput beams.

The beam splitter 240 may split or separate the intensity evenly betweenthe resulting beams of light, or may split them in different proportionsof intensity. In the embodiment shown, the beam splitter 240 is a plate,and the first beam 213 reflects off of the beam splitter 240 while thesecond beam 214 passes through the beam splitter 240. The beam splitter240 may include a coating and/or a filter (e.g., a linear variablefilter) such that one end/side of the beam splitter 240 may havedifferent properties than the opposing end/side. The first beam 213 mayinclude from about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, or 60%)of the optical power of the beam 212, and the second beam 214 mayinclude from about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, or 60%)of the optical power of the beam 212. Thus, in certain embodiments, thefirst beam 213 and the second beam 214 may split the optical power ofbeam 212 evenly (50% for the first beam 213 and 50% for the second beam214). In other embodiments, the first beam 213 may have a greater orlesser percentage than second beam 214 of the optical power of beam 212.An angle between a center of the first beam 213 and a center of thesecond beam 214 may be from about 62° to about 68°, about 70° to about90°, or about 90° to about 110°. The first beam 213 may reflect off ofthe back mirror 260 producing a reflected first beam 215 thatilluminates the imaging target 112 on the imaging surface 110. Thesecond beam 214 may reflect off of the front mirror 262 producing areflected second beam 216 that illuminates the imaging target 112 on theimaging surface 110. An angle between a center of the reflected firstbeam 215 and a center of the reflected second beam 216 may be from about80° to about 100°, about 106° to about 114°, or about 120° to about140°. Although not shown, in at least one embodiment, the second beam214 may illuminate the imaging target 112 on the imaging surface 110directly without reflecting off of the front mirror 262 and producingthe reflected second beam 216.

The reflected first beam 215 and the reflected second beam 216 mayprovide off-axis illumination of the imaging target 112 on the imagingsurface 110. More particularly, the reflected first beam 215 and thereflected second beam 216 may provide substantially symmetricalillumination of the imaging target 112 on the imaging surface 110. Forexample, an angle between the reflected first beam 215 and the imagingsurface 110 may be within +/−10° of the angle between the reflectedsecond beam 216 and the imaging surface 110. A distance from the beamsplitter 240 to the back mirror 260 to the imaging surface 110 may besubstantially equal to (e.g., within 10% of) a distance from the beamsplitter 240 to the front mirror 262 to the imaging surface 110. In atleast one embodiment, the back mirror 260 and/or the front mirror 262may be moved in combination with rotation of beam splitter 240 in orderto vary the illumination of the imaging target 112 on the imagingsurface 110.

FIG. 12 illustrates a simplified schematic top view of a beam of light1212 being emitted from an illumination module 1200 with additionalmirrors 1261-1265, according to an embodiment. In the embodiment shown,the beam of light 1212 may be emitted from the light source of theillumination module 1200 and may be split into a first beam 1213 and asecond beam 1214 by the beam splitter 1240. The first beam 1213 mayreflect off of the beam splitter 1240, and the second beam 1214 may passthrough the beam splitter 1240. The first beam 1213 may reflect off of afirst mirror 1261 and a second mirror 1262 before illuminating theimaging target 112 on the imaging surface 110. The second beam 1214 mayreflect off of a third mirror 1263, a fourth mirror 1264, and a fifthmirror 1265 before illuminating the imaging target 112 on the imagingsurface 110. As with the embodiment in FIG. 11 , the beams 1213, 1214may provide off-axis illumination of the imaging target 112 on theimaging surface 110. In addition, the beams 1213, 1214 may providesubstantially symmetrical illumination of the imaging target 112 on theimaging surface 110.

FIG. 13 illustrates a simplified schematic top view of a beam of light1312 being emitted from an illumination module 1300 with two beamsplitters 1340, 1342, according to an embodiment. In the embodimentshown, the beam of light 1312 may be emitted from the light source ofthe illumination module 1300 and may be split into a first beam 1313 anda second beam 1314 by the first beam splitter 1340. The first beam 1313may reflect off of the first beam splitter 1340 while the second beam1314 may pass through the first beam splitter 1340. In at least oneembodiment, the first beam 1313 may include from about 15% to about 35%(e.g., 15%, 20%, 25%, 30%, or 35%) of the optical power of the beam1312, and the second beam 1314 may include from about 65% to about 85%(e.g., 65%, 70%, 75%, 80%, or 85%) of the optical power of the beam1312.

The first beam 1313 may then reflect off of a first mirror 1360producing a reflected first beam 1315 that illuminates the imagingtarget 112 on the imaging surface 110. The second beam 1314 may be splitinto a third beam 1316 and a fourth beam 1317 by the second beamsplitter 1342. The third beam 1316 may reflect off of the second beamsplitter 1342 while the fourth beam 1317 may pass through the secondbeam splitter 1342. In at least one embodiment, the third beam 1316 mayinclude from about 20% to about 40% (e.g., 33%) of the optical power ofthe second beam 1314, and the fourth beam 1317 may include from about60% to about 80% (e.g., 66%) of the optical power of the second beam1314. The third beam 1316 may then reflect off of a second mirror 1362producing a reflected third beam 1318 that illuminates the imagingtarget 112 on the imaging surface 110. As with the embodiment in FIG. 11, the beams 1315, 1318 may provide off-axis illumination of the imagingtarget 112 on the imaging surface 110. In addition, the beams 1315, 1318may provide substantially symmetrical illumination of the imaging target112 on the imaging surface 110.

The fourth beam 1317 may also illuminate the imaging target 112 on theimaging surface 110. As shown, the fourth beam 1317 may not reflect offof a mirror before illuminating the imaging target 112 on the imagingsurface 110. In an embodiment, an angle between the first beam 1313 andthe fourth beam 1317 may be within about 10° to about 40° of an anglebetween the third beam 1316 and the fourth beam 1317. Similarly, anangle between the reflected first beam 1315 and the fourth beam 1317 maybe within about 10° to about 40° of an angle between the reflected thirdbeam 1318 and the fourth beam 1317.

Although FIG. 13 shows three beams 1315, 1317, 1318 that illuminate theimaging target 112 on the imaging surface 110, in another embodiment,four or more beams may illuminate the imaging target 112 on the imagingsurface 110. For example, four beams may illuminate the imaging target112 on the imaging surface 110 from the front, back, left, and right.

FIG. 14 illustrates a cross-sectional side view of the beam of light 212from FIG. 9 passing through an aperture 1418 in a beam shaper 1410before reaching the beam splitter 240, according to an embodiment. Thebeam shaper 1410 may include one or more lenses (three are shown: 1412,1414, 1416). As shown, the aperture 1418 may be positioned between thesecond and third lenses 1414, 1416; however, in other embodiments, theaperture 1418 may be positioned anywhere within the beam shaper 1410 oralternatively outside of the beam shaper 1410, but before the lightreaches the beam splitter. The size (e.g., cross-sectional area ordiameter) of the aperture 1418 may be fixed. In another embodiment, thesize of the aperture 1418 may be varied to vary the size (e.g.,cross-sectional area or diameter) of the illumination of the imagingtarget 112 on the imaging surface 110. The intensity of the beam oflight 212 from the light source may also be varied to vary the intensityof the illumination of the imaging target 112 on the imaging surface110.

Flat Fielding Calibration

An imager or an imaging system of the present disclosure can be used toimage a variety biological molecules and biological samples such asproteins, peptides, glycoproteins, modified proteins, nucleic acids,DNA, RNA, carbohydrates, lipids, lipidoglycans, biopolymers and othermetabolites generated from cells and tissues. A biological sample can beimaged alone or can be imaged while is it in a membrane, a gel, a filterpaper, slide glass, microplate, or a matrix, such as a polyacrylamidegel or nitrocellulose or PDVF membrane blot, an agarose gel, an agarplate, a cell culture plate or a tissue section slide.

Imaging systems of the present disclosure can image biomolecules andbiological samples in several imaging modes including fluorescentimaging, chemiluminescent imaging, bioluminescent imaging,transillumination or reflective light imaging. In some imaging modes, asample emits light or displays a change in the light it emits(wavelength, frequency or intensity change), without externalillumination or excitation, which can be imaged. In some imaging modes,a sample emits light or has a change in the light it emits (wavelength,frequency or intensity change), following exposure to externalillumination or excitation, which can be imaged. In some imaging modes,a sample reflect light or has a change in the light it reflects(frequency or intensity change), following exposure to externalillumination, which can be imaged.

A common problem faced during imaging (irrespective of imaging mode) isthat when identical samples are placed at different locations of animaging surface or a field of view, the image appears to be non-uniformbased on the location. An imaging surface is exemplified in oneembodiment by part 110 in FIG. 1 , and is also referred to alternativelyherein as imaging area, field of view, a sample screen or a sample tray.In some embodiments, image non-uniformity is displayed as images withsignals of varying intensity for an identical signal measured atdifferent locations on an imaging surface or field of view. In someembodiments, image non-uniformity is displayed as images with differentsignals levels for an identical signal measured at different locationson an imaging surface or field of view. Non-uniformity of image signaldue to location is partially due to one characteristic of an imaginglens i.e., relative illumination.

Non-uniformity of image based on location of sample on an imagingsurface prevents accurate quantitative measurements of biomolecules. Thepresent disclosure describes systems, algorithms and methods used tocalibrate lens assemblies of an imaging system to removenon-uniformities exhibited by the lens to obtain accurate data from thesample images of biomolecules. Calibration of lens assemblies by methodsand systems of the present disclosure removes non-uniformities exhibitedby the lens to obtain accurate data from sample images.

As described in embodiments above, imaging and illuminating devices andsystems of the present disclosure provide imaging correction features toenhance data accuracy with image analysis. These features alone orcombined further with methods, and systems to calibrate imagenon-uniformity to provide superior and accurate quantitativemeasurements of biological samples in an electrophoresis gel or on amembrane and subsequently provide confidence in data analysis andinformation of the image that is obtained from a sample.

In one embodiment, a method for generating an image corrected for anon-uniformity comprises: calculating a relative illumination of animaging lens for a plurality of pixels on an imaging sensor; generatinga flat fielding matrix based upon the relative illumination; capturingan image of one or more biological samples, wherein the image has anon-uniformity; and adjusting the captured image with the flat fieldingmatrix to generate an image corrected for the non-uniformity.

In one embodiment, adjusting the captured image with the flat fieldingmatrix comprises multiplying a captured image of a biological sample bythe value of the flat fielding matrix on a pixel-to-pixel basis togenerate a flat fielded image.

In some embodiments, a method for generating an image corrected for anon-uniformity, can comprise: calculating a relative illumination of animaging lens of a plurality of pixels on an imaging sensor; invertingthe relative illumination to generate a flat fielding matrix; providingthe flat fielding matrix to a user; wherein the user can multiply acaptured image of a biological sample by the value of the flat fieldingmatrix on a pixel-to-pixel basis to generate a flat fielded image. Auser can obtain a captured image using the imager prior to flat fieldingthe image using the flat-fielding matrix. In some embodiments, a usercan choose to generate the image corrected for non-uniformity. In someembodiments, a user can be instructed to generate the image correctedfor non-uniformity by providing the user with a pre-calculated value ofa flat fielding matrix and instructing the user to multiply a capturedimage of a biological sample by the value of the flat fielding matrix ona pixel-to-pixel basis to generate a flat fielded image. In someembodiments, an imaging device or imaging software manufacturer canprovide a flat fielding matrix to a user. In some embodiments, a usercan calculate the value of a flat-fielding matrix using the presentmethods and algorithms.

In one embodiment, a method for generating an image corrected for anon-uniformity, comprising: calculating a relative illumination of animaging lens of a plurality of pixels on an imaging sensor; invertingthe relative illumination to generate a flat fielding matrix. Forexample an imaging device manufacturer or user can generate a flatfielding matrix for a future use.

A flat fielded image, obtained by methods of the present disclosure,displays a correct ratio of a signal level of each captured image of thebiological sample irrespective of its location on an imaging surface,imaging area or field of view. A flat fielded image is an image that hasbeen corrected for a non-uniformity.

Example of Flat Fielding Calibration

One exemplary application mode for an imager or an imaging system of thepresent disclosure is use as an imager for imaging biomolecules (e.g.,but not limited to proteins and nucleic acids in gels or blots) in achemiluminescence mode where the chemiluminescence sample emits lightwithout external illumination and excitation. As noted in sectionsabove, one problem faced while performing chemiluminescence imaging isnon-uniformity of image, wherein when a chemiluminescence sample isplaced at different locations of an imaging surface (such as, part 110in FIG. 1 , an imaging area, a field of view, a sample screen or sampletray), the image signal is different, even for the same sample (such asthe same protein or nucleic band in a gel or a blot).

This problem is illustrated by using a luminometer reference microplate.In one example, FIG. 15 illustrates a perspective view of a luminometerreference plate 1500. However, any luminometer reference plate known inthe art may be used to illustrate this problem. Luminometer referenceplate 1500 has one or more radiation spots. Eight radiation spots areshown on luminometer 1500 numbered 1501-1508. Radiation was blocked fromseven of the radiation spots (e.g., 1501-1507). Only one (e.g., thebrightest) spot 1508 was imaged on different locations of the imagingsurface (on the diagonal of the imaging screen). Spot 1508 ofluminometer reference plate 1500 was placed at different locations onthe field of view (or sample screen). Images were taken of spot 1508 ofluminometer 1500 with a constant exposure time on various parts of theimaging surface and the images were stacked.

FIG. 16A illustrates the stacked images 1600 of spot 1508 of luminometer1500. FIG. 16B illustrates a graph 1610 showing the signal of spot 1508taken at various locations on the imaging surface of an imager of thepresent disclosure, according to an embodiment. Graph 1610 of FIG. 16Bshows that the signals from the same spot 1508 appear to be of differentintensities at different locations on the imaging screen, even thoughall signals are identical since they are emitted by the same emittingspot 1508 on luminometer 1500. As shown in FIGS. 16A and 16B, identicalsignals from a chemiluminescence sample appear to be different due tobeing imaged on different locations of an imaging screen/sample screen.This signal difference due to location is due to a characteristic of animaging lens i.e., relative illumination. In view of the non-uniformityof images, it is not possible for a user to determine if differences inimaging are due to differences in concentration of a biomolecule(protein, nucleic acid, DNA, etc.,) in a band of a luminescent sample orif signal differences are due to the sample band being imaged at adifferent location on sample screen. Hence, reliable and accuratequantitative information cannot be obtained by current imaging methods.

The signal difference described above can be due to relativeillumination of the imaging lens in an imaging system of the presentdisclosure. Relative illumination is a way of representing the combinedeffect of vignetting and roll-off in an imaging lens, and is generallygiven as a percentage of illumination at any point on the sensor,normalized to the position in the field with maximum illumination.Vignetting and roll-off are two separate components of relativeillumination. Because the signal difference described above is due tothe relative illumination (i.e., one of the characteristics of theimaging lens), the difference can be corrected if the relativeillumination is known. The correction process involves creating flatfielding master matrix file based on relative illumination data ofimaging lens, normalizing the flat field master matrix based on themaximum value in the matrix and applying this flat fielding (FF) mastermatrix to a captured image from the system.

FIG. 17 illustrates a graph 1700 showing relative illumination of animaging lens of the present disclosure, according to one embodiment. Theequation for the curve in the graph 1700 can be obtained through alinear or non-linear curve fitting regression. With discrete datapoints, a regression method is applied to fit the curve to a series ofpoints in order to find the best fit equation. Then, the relativeillumination number can be calculated at any position on imaging sensorusing the identified equation. In one embodiment of the presentdisclosure, an algorithm of flat fielding includes the following steps:

-   -   Step 1—Calculating the relative illumination number of all        pixels on the imaging sensor based on the equation identified        from a curve fit regression;    -   Step 2—Inverting the numbers in step 1 and normalize the        inverted numbers based on the maximum value among the inverted        numbers to generate flat fielding master matrix; and    -   Step 3—Upon image acquisition, multiplying the captured image of        a biological sample by the value in the matrix created in step 2        on a pixel-to-pixel basis to generate a final image applied with        flat fielding.

The image after flat fielding shows the correct ratio of signal level ofall bands in a sample.

FIG. 18A illustrates image 1800 from FIG. 16A after flat fielding, andFIG. 18B illustrates a graph 1810 showing a ratio of intensity of spotsover the central spot in FIG. 18A, according to an embodiment. In otherwords, FIG. 18B shows the relative percentage of those spots before andafter flat fielding. As shown, the ratio of individual spot intensityover the spot with maximum intensity value (FIG. 18A) before applyingflat fielding can be about 0.7, whereas the ratio is increased to begreater than 0.95 after flat fielding. As such, there is significantintensity compensation after flat fielding application.

FIG. 19A illustrates an image 1900 of a chemiluminescence sample at 1×zoom level at a middle position of the field of view, FIG. 19Cillustrates an image 1910 of the chemiluminescence sample at 1× zoomlevel at a top right position of the field of view, and FIG. 19Billustrates an image 1920 of the chemiluminescence sample at 1× zoomlevel at a position between the middle and the top right positions ofthe field of view, according to an embodiment.

FIG. 20A illustrates an image 2000 showing two rows of bands that arequantified, FIG. 20B illustrates a graph 2010 showing the intensity ofthe bands on the first row before flat fielding, FIG. 20C illustrates agraph 2020 showing the intensity of the bands on the first row afterflat fielding, FIG. 20D illustrates a graph 2030 showing the intensityof the bands on the second row before flat fielding, and FIG. 20Eillustrates a graph 2040 showing the intensity of the bands on thesecond row after flat fielding, according to an embodiment. As seen, theintensity of the individual band changes with the location of sample onthe imaging surface (with identical sample), whereas, after flatfielding, the intensity does not vary with location on the imagingsurface.

Generating a Flat Fielding (FF) Master

FIG. 21A illustrates Table 2100 showing relative illumination of imaginglens against image height on a detection sensor with a 1× zoom, and FIG.21B illustrates Table 2110 showing relative illumination of a CCD sensorimaging lens against image height with a 2× zoom, according to anembodiment.

FIG. 22 illustrates a plot 2200 showing that relative illumination issymmetrical with respect to the center of the image sensor, according toan embodiment. The maximum image height is about 8 mm. Simulations wererun from 0 mm to 8 mm.

FIG. 23A illustrates a graph 2300 showing a best fit curve with a 1×zoom, and FIG. 23B illustrates a graph 2310 showing a best fit curvewith a 2× zoom, according to an embodiment. The curves may be a firstdegree polynomial, a second degree polynomial, a third degreepolynomial, or the like. Image height is calculated from this equation:h=√{square root over ((x−x _(c))²+(y−y _(c))²)}×pixel heightwhere h represents the height (in mm) from the center pixel of thedetection sensor, x_(c) represents the x-coordinate of the center pixel,and y_(c) represents the y-coordinate of the center pixel. The pixelheight in this example is 3.69 μm/pixel. Given this, for a 1× zoom,relative illumination can be calculated from the equation of the bestfit curve:RI=−0.3654h ²−3.1275h+100.15Where RI represents the relative illumination (%), 0≤RI≤100.For Bin 1×1 image:

-   -   Width=3360 pixels→x_(c)=1690    -   Height=2704 pixels→y_(c)=1352    -   For pixel (1,1)        h=√{square root over ((1−1690)²+(1−1352)²)}×3.69 μm=7.98 mm.        RI=−0.3654×7.98092²−3.1275×7.98092+100.15=51.9%

FIG. 24 illustrates a simulation image 2400 at a 1× zoom, according toan embodiment.

FIG. 25 illustrates a flat fielding master image 2500, according to anembodiment. The value of each pixel in the master image may equal RI⁻¹.

Application of Flat Fielding Master Images

FIG. 26A illustrates an image 2600 with a sample at a middle position onthe field of view with a 1× zoom, FIG. 26C illustrates an image 2610with the sample at a top right position of the field of view with a 1×zoom, and FIG. 26B illustrates an image 2620 with the sample between themiddle and top right positions in the field view, according to anembodiment. In this example, the sample is a western blot membrane withequal amount of protein visualized with chemiluminescent substrate, thebin number is 5, the zoom may be 1× or 2×, the gain is high (e.g., 55),and the expiration time is 60 seconds.

The flat field master matrix has been applied to the images in FIGS.26A-26C. Rectangular masks were drawn over selected bands, and the meanpixel intensity was measured. A macro was used to ensure that themeasured area selected for each band is at the same position for theimages before applying flat fielding master matrix and after applyingflat fielding master matrix. Although the western blot membrane wasprepared with samples having equal protein amount, there was still avariance in signal value between different bands. For each individualband, the signal intensity should be similar in the same row regardlessof membrane position.

FIG. 27 illustrates an image 2700 showing two rows of eight sample bandseach, according to an embodiment.

FIG. 28A illustrates a graph 2800 showing the first row before flatfielding, FIG. 28B illustrates a graph 2810 showing the first row afterapplying flat fielding master matrix, FIG. 28C illustrates a graph 2820showing the second row before applying flat fielding master matrix, andFIG. 28D illustrates a graph 2830 showing the second row after applyingflat fielding matrix, according to an embodiment. The zoom is 1× inFIGS. 28A-28D. Before applying flat fielding master matrix, thedifference in the ADU (analog-to-digital unit) value is due to theposition of the membrane on imager surface/imager screen. After applyingflat fielding master matrix, the ADU values are similar regardless ofthe position of the membrane.

FIG. 29A illustrates a graph 2900 showing the first row before applyingflat fielding master matrix, FIG. 29B illustrates a graph 2910 showingthe first row after applying flat fielding master matrix, FIG. 29Cillustrates a graph 2920 showing the second row before applying flatfielding master matrix, and FIG. 29D illustrates a graph 2930 showingthe second row after applying flat fielding master matrix, according toan embodiment. The zoom is 2× in FIGS. 29A-29D.

FIG. 30 illustrates a chart 3000 showing the membrane position (e.g.,middle, top right location/position of the membrane and its sample bandson an imager surface/imager screen) before and after applying flatfielding master matrix, according to an embodiment. As shown, the bandsare fainter at the top right position before applying flat fieldingmaster matrix, and the bands have similar brightness after applying flatfielding master matrix.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all sub-ranges subsumedtherein. For example, a range of “less than 10” can include any and allsub-ranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all sub-ranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5. In certain cases, the numerical values asstated for the parameter can take on negative values. In this case, theexample value of range stated as “less than 10” can assume negativevalues, e.g. −1, −2, −3, −10, −20, −30, etc.

While the teachings have been described with reference to the exemplaryembodiments thereof, those skilled in the art will be able to makevarious modifications to the described embodiments without departingfrom the true spirit and scope. The terms and descriptions used hereinare set forth by way of illustration only and are not meant aslimitations. In particular, although the method has been described byexamples, the steps of the method may be performed in a different orderthan illustrated or simultaneously. Furthermore, to the extent that theterms “including”, “includes”, “having”, “has”, “with”, or variantsthereof are used in either the detailed description and the claims, suchterms are intended to be inclusive in a manner similar to the term“comprising.” As used herein, the term “one or more of” with respect toa listing of items such as, for example, A and B, means A alone, Balone, or A and B. Those skilled in the art will recognize that theseand other variations are possible within the spirit and scope as definedin the following claims and their equivalents.

Other embodiments of the disclosure will be apparent to those skilled inthe art from consideration of the specification and practice of thedisclosure disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the disclosure being indicated by the following claims.

As used herein, the terms “inner” and “outer”; “up” and “down”; “upper”and “lower”; “upward” and “downward”; “above” and “below”; “inward” and“outward”; and other like terms as used herein refer to relativepositions to one another and are not intended to denote a particulardirection or spatial orientation. The terms “couple,” “coupled,”“connect,” “connection,” “connected,” “in connection with,” and“connecting” refer to “in direct connection with” or “in connection withvia one or more intermediate elements or members.”

What is claimed is:
 1. An illumination system, comprising: a surfaceconfigured to have an imaging target placed thereon; a light sourceconfigured to emit a beam of light; a first beam splitter configured tosplit the beam of light directly emitted from the light source into afirst beam and a second beam; a first mirror configured to reflect thefirst beam to provide a reflected first beam that illuminates thesurface; and a second mirror configured to reflect the second beam toprovide a reflected second beam that illuminates the surface, a secondbeam splitter configured to split the reflected first beam into tworeflected beams that provide different degrees of off-axis illuminationof the surface.
 2. The illumination system of claim 1, wherein thereflected first beam and the reflected second beam provide off-axisillumination of the surface.
 3. The illumination system of claim 1,wherein the reflected first beam and the reflected second beam providesubstantially symmetrical illumination of the surface.
 4. Theillumination system of claim 1, wherein the beam of light has a beam oflight optical power, the first beam has a first beam optical power andthe second beam has a second beam optical power, and wherein the firstbeam optical power and the second beam optical power are each at least40% of the beam of the light optical power.
 5. The illumination systemof claim 4, wherein the first beam optical power and the second beamoptical power are each at least 45% of the beam of the light opticalpower.
 6. The illumination system of claim 5, wherein the first beamoptical power and the second beam optical power are substantially equal.7. The illumination system of claim 1, further comprising a thirdmirror, wherein the reflected first beam or the reflected second beam isconfigured to reflect off of the third mirror prior to illuminating thesurface.
 8. The illumination system of claim 1, wherein the beamsplitter is configured to split the beam of light from the light sourceinto the first beam, the second beam, and a third beam.
 9. Theillumination system of claim 1, wherein the beam splitter comprises aprism, a plate, a dielectric mirror, a metal coated mirror, a beamsplitter cube, a fiber optic beam splitter, or optical fibers configuredto collimate light into a bundle before producing two or more outputbeams.
 10. The illumination system of claim 1, wherein the first beamreflects off of the beam splitter, and wherein the second beam passesthrough the beam splitter.
 11. The illumination system of claim 1,further comprising a third beam splitter configured to split thereflected second beam into two reflected beams that provide differentdegrees of off-axis illumination of the surface.
 12. The illuminationsystem of claim 1, wherein an angle between a center of the first beamand a center of the second beam is from about 62° to about 68°.
 13. Theillumination system of claim 1, wherein an angle between a center of thereflected first beam and a center of the reflected second beam is fromabout 106° to about 114°.
 14. The illumination system of claim 1,wherein a first distance from the beam splitter to the first mirror tothe surface is substantially equal to a second distance from the beamsplitter to the second mirror to the surface.
 15. An illuminationsystem, comprising: a surface configured to have an imaging targetplaced thereon; a light source configured to emit a beam of light; abeam splitter configured to split the beam of light directly emittedfrom the light source into a first beam and a second beam; a firstmirror configured to reflect the first beam from the beam splitter toprovide a reflected first beam that illuminates the surface; a secondmirror configured to reflect the second beam to provide a reflectedsecond beam that illuminates the surface; and a second beam splitterconfigured to split the reflected first beam into two reflected beamsthat provide different degrees of off-axis illumination of the surface,wherein the beam of light has a beam of light optical power, the firstbeam has a first beam optical power and the second beam has a secondbeam optical power, and wherein the first beam optical power and thesecond beam optical power are each at least 40% of the beam of the lightoptical power.
 16. An illumination method, comprising: providing asurface with an imaging target placed thereon; providing a beam of lightfrom a light source; splitting the beam of light from the light sourceinto a first beam and a second beam; and illuminating the surface,wherein illuminating comprises: (i) using a first mirror to reflect thefirst beam to produce a reflected first beam that illuminates thesurface, and (ii) using a second mirror to reflect the second beam toproduce a reflected second beam that illuminates the surface, and (iii)splitting the reflected first beam into two reflected beams that providedifferent degrees of off-axis illumination of the surface.
 17. Themethod of claim 16, wherein the reflected first beam and the reflectedsecond beam provide off-axis illumination of the surface.
 18. The methodof claim 16, wherein the reflected first beam and the reflected secondbeam provide substantially symmetrical illumination of the surface. 19.The method of claim 16, wherein the beam of light has a beam of lightoptical power, the first beam has a first beam optical power and thesecond beam has a second beam optical power, and wherein the first beamoptical power and the second beam optical power are each at least 40% ofthe beam of the light optical power.
 20. An illumination system,comprising: a surface configured to have an imaging target placedthereon; a light source configured to emit a beam of light; a first beamsplitter configured to split the beam of light from the light sourceinto a first beam and a second beam; a first mirror configured toreflect the first beam to provide a reflected first beam thatilluminates the surface; a second mirror configured to reflect thesecond beam to provide a reflected second beam that illuminates thesurface; a second beam splitter configured to split the reflected firstbeam into two reflected beams that provide different degrees of off-axisillumination of the surface; and a third beam splitter configured tosplit the reflected second beam into two reflected beams that providedifferent degrees of off-axis illumination of the surface.